Table 2.

Immunohistochemical staining with polyclonal antibodies applied on porcine lung tissue.

	Antibody and specificity
	Rabbit anti-human lysozyme	Goat anti-pig IgM μ-chain	Goat anti-pig IgG-Fc fragment	Goat anti-pig IgA
Source	Dakocytomation	Bethyl Laboratories	Bethyl Laboratories	Bethyl Laboratories
Dilution	1:350	1:5000	1:7000	1:4000
Fixation of tissue	Formalin	Formalin	Formalin	Formalin
Blocking of unspecific protein binding	1% blocking reagent	5% rabbit serum	5% rabbit serum	5% rabbit serum
Antigen retrieval	Trypsin (1 mg/mL)	Protease (0.36 mg/mL)	Protease (0.36 mg/mL)	Protease (0.36 mg/mL)
Detection	EnVision-HRP	PAP-goat	PAP-goat	PAP-goat
Chromogene	DAB/10 min	DAB/10 min	DAB/10 min	DAB/10 min

Source: Dakocytomation, Glostrup, Denmark. Bethyl Laboratories, Montgomery, TX, USA.

Blocking of unspecific protein binding. Blocking reagent (Boehringer, Mannheim, Germany). Rabbit serum (X0902, Dakocytomation).

Antigen retrieval. Trypsin (Sigma-Aldrich, Brøndby, Denmark) (1 mg/mL) in Tris-buffered saline (TBS) for 2 h followed by incubation in icecold TBS (pH 7.4) for 5 min. Protease (Sigma-Aldrich) (0.36 mg/mL) in TBS for 5 min followed by incubation in icecold TBS (pH 7.4) for 5 min.

Detection. EnVision-HRP (K4002, Dakocytomation). For all horseradish peroxidase (HRP)-based detection systems endogenous tissue hydrogen peroxide was quenched with 0.6%/3% H₂O₂ in TBS (pH 7.4) for 20 min before blocking of unspecific protein binding. PAP-goat (B0157, Dakocytomation) 1:100 in TBS after previous incubation in polyclonal rabbit anti-goat (Z0454, Dakocytomation) 1:200 in TBS. Chromogene. DAB (KemEnTec; Copenhagen, Denmark).